Urokinase (UK), streptokinase (SK) and tissue plasminogen activator (t-PA) are currently used as thrombolytic agents.
The primary structure of human urokinase has been described by Heynecker et al. (JP-A-59-51300, the term "JP-A" used herein means "an unexamined published Japanese patent application") and the folded structure thereof has been described as well as shown in FIG. 1. This folded structure, which has been proposed based on homology to other proteins, can be divided into the following three domains. The first domain on the N-terminal side is a domain homologous to epidermal growth factor. Hereinafter, this domain is referred to as "growth factor domain". The second domain is the so-called "kringle domain". The third domain at the C terminal is the so-called serine protease domain. Hereinafter, the third domain is referred to as "protease domain".
Human urokinase occurs in two forms, a single-chain form and a double-chain form. The single chain form of UK is called prourokinase (hereinafter referred to as "pro-UK") and, in such a structure, it has no thrombolytic activity. Only upon cleavage by plasmin into a double-chain structure, can it exhibit thrombolytic activity. However, when pro-UK is cleaved by thrombin, which cleaves pro-UK at a site two amino acids upstream from the plasmin cleavage site, the resulting double-chain structure has no thrombolytic activity any longer [Ichinose et al., J. Biol. Chem., 261, 3486 (1986)]. Thus, after administration into the blood, pro-UK is cleaved by thrombin which is present in the blood into an inactive double-chain structure. Furthermore, if the purification process for pro-UK is contaminated by a thrombin-like protease, the protease will possibly cleave pro-UK into an inactive double-chain structure.
To prevent pro-UK from being cleaved by a thrombin-like protease, a pro-UK derivative resulting from the introduction of a substitute amino acid into the thrombin cleavage site at position P.sub.1 or P.sub.1 ', has been proposed (EP-A-0200451 and JP-A-62-143686).
However, since the thrombin cleavage site is separated by only two amino acids from the plasmin cleavage site, the substitute amino acid introduced in the vicinity of the plasmin cleavage site namely at P.sub.3 to P.sub.1 ', simultaneously makes it difficult for plasmin cleavage to occur, hence the conversion of the pro-UK derivative obtained into an active form becomes difficult and a decreased specific activity results. In fact, with the pro-UK derivative mentioned above, in which the position of amino acid substitution is P.sub.3 or P.sub.2 relative to the plasmin cleavage site, a decrease in susceptibility to plasmin, hence a decrease in specific activity, has been observed.
As regards the numbering of the amino acid residues of the substrate peptide to be cleaved, the peptide bond to be cleaved is represented by --P.sub.1 --P.sub.1 '-and the amino acids on the carboxyl side to the peptide bond to be cleaved are given position codes P.sub.1, P.sub.2, P.sub.3, P.sub.4 etc. from the amino acid constituting the carboxyl side of said peptide bond toward the amino terminus. The amino acids on the amino side of this bond are given position codes P.sub.1 ', P.sub.2 ', P.sub.3 ', P.sub.4 ' etc. from the amino acid constituting the amino side of said peptide bond toward the carboxyl terminus.
It is an object of the invention to develop a novel pro-UK derivative which is stable against any thrombin-like protease capable of inactivating pro-UK and shows no substantial decrease in specific activity.
Heretofore, reports have been published on the construction of pro-UK derivatives, which are more or less resistant to cleavage by thrombin, by introducing a substitute amino acid in position P.sub.1 with respect to the thrombin cleavage site (position P.sub.3 with respect to the plasmin cleavage site; 156th amino acid of mature pro-UK) or position P.sub.1 ' with respect to the thrombin cleavage site (P.sub.2 with respect to the plasmin cleavage site; 157th amino acid of mature pro-UK). However, since the position of amino acid substitution in these derivatives is only two or three amino acids away from the plasmin cleavage site, the pro-UK derivatives may possibly have a decreased specific activity as a result of a decrease in susceptibility to plasmin. In fact, the present inventors have confirmed that amino acid substitution in mature pro-UK at position 157 (P.sub.2 with respect to the plasmin cleavage site) results in a decrease in specific activity. Furthermore, in the case of amino acid substitution at position 157, it has been found that there is another problem in addition to specific activity decrease. Namely, the resistance of the pro-UK derivatives to thrombin is still unsatisfactory.